(a) Field of the Invention
The present invention relates to agonists and antagonists of gonadotropin-releasing hormone-2 (GnRH-2) which regulate the activity of GnRH-2 by specifically binding to GnRH-2 receptors, and pharmaceutical compositions for regulating the GnRH-2 receptors.
(b) Description of the Related Art
Gonadotropin-releasing hormone (GnRH) is a neuropeptide having an important role in reproductive physiology, and it is widely used as a treatment agent for endocrinal and neuroendocrinal diseases (Sealfon S C, et al., Endocr. Rev. vol.18 pp. 180-205, 1997). Thus far, about 14 forms of GnRH have been found in 70 different species of vertebrates and invertebrates, and two or more GnRHs exist in each individual (Fernald R D, et al., Front Neuroendocrinol, vol.20, pp. 224-240, 1999).
The first type of GnRH (GnRH-1) is a hypothalamic GnRH. The neurons of GnRH-1 are mostly distributed in the hypothalamus, and their nerve terminals exist at the median eminence. GnRH-1 is secreted at the median eminence, arrives at the pituitary gland via a portal system that connects the hypothalamus and pituitary gland, and regulates the functions of the pituitary gland. GnRH-1 regulates the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH), and LH and FSH regulate gonadal development and steroid synthesis. Therefore, GnRH-1 is deeply involved in the regulation of reproductive endocrinology in vertebrates (Seeburg P H, et al., Recent Prog Horm Res vol.43 pp. 69-98, 1986).
The typical GnRH-1 is mammalian GnRH (mGnRH) that was found for the first time in the mammalian hypothalamus (Matsuo H, et al., Biochem Biophys Res Commun vol.43 pp. 1334-1339, 1971; Burgus R, et al., Proc Natl Acad Sci USA vol.69 pp. 278-282, 1972). mGnRH is found in most vertebrates including Aves, Reptilia, and Amphibia, as well as Mammalia. However, in fish several birds, and amphibians, different forms thereof are found (Miyamoto K, et al., Life Sci vol. 32, pp. 1341-1347, 1983; Powell J F et al., Proc Natl Acad Sci USA vol. 91 pp. 12081-12085, 1994; Yoo M S, et al., Mol Cell Endocrinol vol.164 pp. 197-204, 2000).
The second type of. GnRH (GnRH-2) is chicken GnRH-II (His5Trp7Tyr8GnRH, cGnRH-II) that was found for the first time in the chick brain (Miyamoto K et al, Proc Natl Acad Sci USA vol.81 pp. 3874-3878). The neurons of cGnRH-II are mostly found in the midbrain, and their nerve terminals exist in the posterior hypothalamus or hindbrain. Although the exact functions of cGnRH-II are not well known, it is postulated that it generally has an important role in functioning as a neuromodulator and in regulating sexual behaviors (Troskie B, et al., Neuroendocrinology vol.65 pp. 396-402, 1997). Further, as several nerve terminals extend to the hypothalamus, it is probably involved the regulation of reproductive physiology. cGnRH-II is found in almost all vertebrates, i.e., Pisces, Amphibia, and Reptilia, as well as Mammalia including humans, and mutant forms thereof have not been found (White R B, et al., Proc Natl Acad Sci USA vol.95 pp. 305-309, 1998).
Hence, in vertebrates higher than Pisces, GnRHs that exist in a single species are cGnRH-II (GnRH-2) and mGnRH (or a mutant form of GnRH-1). Furthermore, GnRH-1 and GnRH-2 have been known to be expressed in peripheral tissues of the body, in addition to the central nervous system. Particularly, the expression of GnRH-1 and GnRH-2 has been reported in tissues regulating the immune and reproductive systems (Kang S K, et al., Endocrinology vol.142 pp. 182-192, 2001; Wilson T M, et al., Mol Endocrinol vol.9 pp. 44-53, 1995; Dong K W, et al., Mol Cell Endocrinol vol.117 pp. 121-130, 1996). Therefore, GnRH is expected to function as a local regulator in immune and reproductive systems, in addition to its functions in the neuroendocrine regulation.
Studies of GnRH have been vigorously conducted, as they provide essential information on the design of clinical treatment agents as well as the understanding of reproductive functions. GnRH is secreted in a pulsatile manner, and such a secretion manner plays an important role in regulating the synthesis and secretion of steroid hormones in the gonads and maintaining normal reproductive functions. Endocrine or neuroendocrine diseases generated by the modification of GnRH genes and dysfunction of GnRH neurons can be cured by treating GnRH in a pulsatile manner. However, if GnRH is treated continuously at a high concentration, the function of GnRH receptors becomes deficient, and ultimately hypofunction of the gonads is induced. By virtue of such GnRH functions, GnRH is used for the treatment of reproductive endocrine diseases and precocious puberty, as well as the control of menstruation periods in in vitro fertilization (Huirne J A, et al., Lancet vol.358 pp. 1793-1803, 2001). Also, GnRH is used for the treatment of cancers that are sensitive to steroid hormones, i.e., prostate cancer, breast cancer, and ovarian cancer (Schally A V, Peptides vol.20 pp. 1247-1262, 1999; Grundker C, et al., Eur J Endocrinol vol.146 pp. 1-14, 2002).
Recent studies have revealed that GnRH-2 is more effective than GnRH-1 for the treatment of some cancer cells, and that signal transduction via GnRH-2 is different from that of GnRH-1 (Kang et al., ibid.; Grundker et al., ibid.). Also, as a receptor sensitive to GnRH-2 was identified in monkeys (Neill J D, et al., Biochem Biophys Res Commun vol. 282 pp. 1012-1018, 2001; Millar R, et al., Proc Natl Acad Sci USA vol. 98 pp. 9636-9641, 2001), studies about the physiological functions of GnRH-2 via such receptor became necessary. Due to the clinical importance of GnRH, approximately 3000 GnRH agonists and antagonists have been developed thus far. Most GnRH agonists and antagonists were developed by partially modifying the amino acid structure of GnRH-1 to increase their binding affinity toward the GnRH receptors and to slow down their in vivo degradation rate, thereby maximizing their efficiency (Sealfon et al., ibid.).
Recently, the inventors cloned three types of receptors that are very sensitive to GnRH-2 in bullfrogs (Wang et al., Proc Natl Acad Sci USA vol. 98 pp. 361-366, 2001). All of the bullfrog GnRH receptors (bfGnRHR) react more sensitively to GnRH-2 than GnRH-1. Also, the bfGnRHR shows great structural differences from the GnRH receptors that were found in mammals, but it is very similar in structural aspects to the GnRH receptors found in nonmammals, i.e., Pisces, Amphibia, and Aves. In addition, it is structurally similar to the receptor sensitive to GnRH-2 that has been recently found in monkeys (monkey GnRHR-2) (Neill et al., ibid.; Millar et al., ibid.). Accordingly, the bfGnRHR is very similar to the second GnRH receptor of mammals in respect of its structure and function.
Thousands of GnRH-1 agonists have been developed because of the reproductive and physiological importance of GnRH-1, as well as its clinical efficacy in reproductive dysfunction and cancer treatment. However, little development and study of GnRH-2 analogs has been conducted, for several reasons. GnRH-2 was characterized relatively later than GnRH-1, and the fact that GnRH-2 is present in most vertebrates including humans was revealed only four years ago. In addition, in vivo functions of GnRH-2 have not been fully known, and the discovery of the GnRH-2 receptor which is the most important factor in the development of GnRH-2 analogs was delayed. In nonmammalian animals, the GnRH-2 receptors were found in catfish in 1997 and goldfish in 1998 (Tensen et al., 1997; Illing et al., 1999), and recently three types of GnRH receptors were found in bullfrogs by the inventors, and thereby studies on them became serious (Wang et al., ibid.). Moreover, in the case of mammals, the fact that the second receptor that is sensitive to GnRH-2 is present in monkeys was found extremely recently (Millar et al., ibid.; Neill et al., ibid.]. Hence, studies regarding the development of GnRH-2 analogs have been delayed.
Several GnRH-2 agonists were developed through the substitution of the 6th amino acid of GnRH-2 ([D-Arg6]GnRH-2, [D-Leu6]GnRH-2, [D-Trp6]GnRH-2, [D-t-bu-Ser6]GnRH-2, Siler-Khodr and Khodr, et al., ibid. and PCT Laid-Open Publication WO No. 01-74377). However, these agonists studies were carried out in cell lines having receptors sensitive to GnRH-1, and they were not conducted with regard to receptors sensitive to GnRH-2.
Therefore, the current circumstance is that the development of GnRH-1 antagonists and studies about GnRH-2 receptors have been carried out, but the development of GnRH-2 agonists and antagonists has not been conducted. Hence, for clinical applications and studies of physiological functions via GnRH-2, the development of GnRH-2 agonists and antagonists is required.